Hipks (Hipk1, Hipk2 and Hipk3) encode a novel family of nuclear homeodomain interacting protein kinases that act as transcriptional co-repressors for homeodomain transcription factors. HIPK proteins contain multiple functional domains, including a protein kinase domain, an interacting domain with homeoproteins, the PEST sequence and the YH domain at the C- terminus with unknown function. HIPKs localize to nuclear speckles, and speckle retention signal (SRS) has been determined using various GFP:HIPK fusion constructs. The SRS follows the interaction domain with homeoproteins. In oreder to explore the possibility that subnuclear localization of HIPKs is regulated for their functions, the yeast two-hybrid screen was performed using the SRS as a bait to find interacting molecules with the SRS. Candidate genes were cloned, and further characterization of these genes in relation to subnuclear locaization of HIPKs is underway. Transgenic flies harboring gene carsettes that produce mRNA for HIPK2 were generated in order to investigate in vivo function of HIPKs. Ectopic expression of HIPK2 throughout mesoderm caused abnormal muscle phenotype which is similar to the phenotype generated by ectopic expression of NK-3, suggesting that HIPK function may be corelated with the activity of homeoproteins that interact with HIPKs. For the study of loss-of-function of HIPKs, genomic DNAs of HIPKs were cloned, and characterization of gene structure and construction of plasmids for knock-out experiments are underway.